US20060233780A1 - Method for treating oncological diseases - Google Patents
Method for treating oncological diseases Download PDFInfo
- Publication number
- US20060233780A1 US20060233780A1 US10/564,861 US56486106A US2006233780A1 US 20060233780 A1 US20060233780 A1 US 20060233780A1 US 56486106 A US56486106 A US 56486106A US 2006233780 A1 US2006233780 A1 US 2006233780A1
- Authority
- US
- United States
- Prior art keywords
- dna
- blood
- agent
- day
- extracellular dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/21—Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
- C12Y301/21001—Deoxyribonuclease I (3.1.21.1)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
Definitions
- the invention is related to medicine and veterinary and can be used for treating in the main of solid tumors.
- Chemotherapy, radiotherapy, biotherapy and more recently the immunotherapy are the most-used non-surgical methods of treating of cancer diseases and its are meant for destruction, damage or inactivation of cancer cell intracellular DNA.
- the chemotherapy approach is based on well known compounds: for platinum preparations see “Molecular mechanisms involved in cisplatin cytotoxicity”. Jordan P, Carmo-Fonseca M, Cell Mol Life Sci August 2000 v 57: pp. 1229-35, for anthracycline antibiotics, see “Daunorubicin and doxorubicin, anthracycline antibiotics, a physicochemical and biological review”, Aubel-Sadron G, Londos-Gagliardi D, Biochimie May 1984 v. 66: pp.
- biotherapeutic and immunotherapeutic methods which meant for induction of apoptosis of cancer cells—the process of death of cancer cell, which starts with the activation of intracellular nucleases with following degradation of intracellular DNA of the tumor cell, for example by means administration of genotherapeutic constructions, which consists of genes inducing apoptosis, see “Phase I trial of adenovirus-mediated p53 gene therapy for recurrent glioma: biological and clinical results” Lang F F, Bruner J M, Fuller G N, Aldape K, Prados M D, Chang S, Berger M S, McDermott M W, Kunwar S M, Junck L R, Chandler W, Zwiebel J A, Kaplan R S, Yung W K, J Clin Oncol July 2003 1 21:13 2508-18, or genes coding the factors which activate the nucleases triggering apoptosis, see “Adenovirus-mediated transfer of caspase-8 augments cell death in gliomas:
- the chemotherapy with the Etopozide-4-Demetilpipodophylotoxe(4,6-O—R)-etiliden)-b-D-glycopiranozid was selected by us as a prototype.
- Topoizomeraze II is essential cell enzyme which regulates many aspects of DNA function. The enzyme is responsible for interconversion of different topological forms of intracellular DNA by means of generation of transitory breaks of double-stranded DNA.
- Etopozide as Topoizomeraze II inhibitor, increases intracellular level of “broken DNA—Topoizomeraze II” complexes.
- Method-prototype as well as any other well-known methods, is their low efficacy. It is explained by following. Method-prototype as well as other known methods implies the cancer cell and mostly the intracellular DNA as a therapeutic target.
- cancer cells become, as a rule no sensitive to the therapy before they are adequately eliminated by means of the using method;
- intracellular DNA is a difficult-to-approach target, and it leads to necessity of high-dosing antineoplastic chemotherapy and/or necessity of complicated delivery systems.
- drug Delivery Systems drug Delivery Systems
- method-prototype which makes provision for influence on intracellular DNA of cancer cells also damages DNA of healthy cells and that's reason of it's high toxicity.
- this problem is resolved by administrating into systemic circulation an agent which destroys blood extracellular DNA; and the said agent can be introduced in the dose which provide the alteration an electrophoretic profile of blood extracellular DNA, which could be detectable by puls-gelelectrophoresis; the agent can be introduced in doses and regime schedule providing a plasma hydrolytic activity, measured in blood plasma which exceeds 150 Kuntz units/liter of blood plasma, and this level can be supported for more than 12 hours within 24 hours in total; the treatment can be carried out continuously no less than 48 hours; DNAase enzyme can be used as agent to destroy extracellular blood DNA; in particular bovine pancreatic DNAase can be introduced parenterally in doses ranged from 50 000 Kunitz units/day till 25 000 000 000 Kunitz units/day per day every day during period of 5-360 days, in particular recombinant human DNAase (dornase—alpha) can be parenterally
- the extracellular blood DNA of cancer patients contains the unique quantitative and qualitative repertoire of genes and regulating genetic elements which great differ from the repertoire of DNA which is described in human genome.
- the extracellular DNA of cancer patients contains mainly unique human genes, including genes which are involved in development and maintenance of malignant behavior of cancer cells.
- the following agents were used which destroys extracellular blood DNA: bovine pancreatic DNAase (Sigma and Samson Med), recombinative human DNAase 1 (Dornase alpha; Genetech), Serratia Mercenses extracellular nuclease.
- bovine pancreatic DNAase Sigma and Samson Med
- recombinative human DNAase 1 (Dornase alpha; Genetech)
- Serratia Mercenses extracellular nuclease.
- the solutions of DNA-se for administration was made by dissolving of mother solutions of DNA-se in sterile phosphate buffer just before administration.
- the volume of DNA solution was 2 ml, volume of each step of CsCl was 1 ml.
- mice Lewis lung carcinoma and Erlich carcinoma were used in experiments. Cells were cultivated in RPMI-1640 medium with 10% calf serum and 1% penicillin-streptomycin in atmosphere of 5% CO2.
- mice For tumors inoculation in mice the cells were cultivated till monolayer is formed, then detached with tripsin-EDTA buffer. The cells were washed 3 times by centrifuging in phosphate buffer and then resuspended up to 0, 5*10 7 /ml concentration in the same buffer. The cell viability was determined with metylene blue staining in haemocitometer. Cells suspensions with no less than 95% of living cell were used for transplantation.
- mice and white randomly breeded mice from “Rappolovo” animal house were used. Weight of animals was 24-26 g. 6-7animals were kept in one cage on a standard diet without limitation of water. LLC cells in dose 5*10 5 per mice in 0,1 ml of phosphate buffer were transplanted into thigh soft tissues. Erlich tumors were transplanted by administration of 0,2 ml of 10% cell suspension in physiological solution.
- DNA was isolated according to aforesaid protocol. The DNA level was measured with PicoGreen kit. Electrophoresis of extracellular blood DNA was performed with 1% agar gel. DNA was visualized with etidium-bromide solution. The levels of high molecular DNA fraction (more than 300 base pairs) were determined by densitometry. Lambda phage DNA, digested with EcoRI and HindIII was used as electrophoresis marker.
- mice bearing Erlich carcinoma was used as control.
- the mice were injected with 0,2 ml of phosphate buffer intraperitoneally twice a day every day from day 3 to day 7 after the tumor cell transplantation.
- mice bearing Erlich carcinoma were introduced with intraperitoneal injections of DNAase in dose of 1 mg/kg of body weight in 0,2 ml of phosphate buffer four times daily every day from day 3 to day 7 after the tumor cell transplantation.
- mice bearing Erlich carcinoma were administered with intraperitoneal injections of DNAase in dose of 0,5 mg/kg of body weight in 0,2 ml of phosphate buffer four times daily every day from day 3 to day 7 after the tumor cell transplantation.
- mice bearing Erlich carcinoma were administered with intraperitoneal injections of DNAase in dose of 0,1 mg/kg of body weight in 0,2 ml of phosphate buffer four times daily every day from day 3 to day 7 after the tumor cell transplantation.
- mice bearing Erlich carcinoma were administered with intraperitoneal injections of DNAase in dose of 0,05 mg/kg of body weight in 0,2 ml of phosphate buffer four times daily every day from day 3 to day 7 after the tumor cell transplantation.
- the results were evaluated as tumor Growth Inhibitory Index(GII) (%) at the last day of DNAase injections.
- the quantification of blood plasma extracellular DNA and it's electrophoretic qualification were also performed.
- Tumor size, extracellular DNA level and extracellular DNA electrophoresis profile on day 7 after tumor transplantation TABLE 1 Presence of high Extracellular molecular fractions Tumor Inhibition DNA level, of extracellular Group volume (%) (ng/ml) DNA Control 98 +/ ⁇ 14 — 104.8 100%* 1 mg/kg ⁇ 23 +/ ⁇ 6 76% 38.3 0 0.5 mg/kg 32 +/ ⁇ 6 67% 55.1 25% 0.1 mg/kg 58 +/ ⁇ 12 37% 78.0 70% 0.05 mg/kg 87 +/ ⁇ 11 10% 98.7 100% *The control group electrophoretic density were considered as 100%.
- mice bearing LLC 5 groups of mice bearing LLC were used.
- mice were administered with intraperitoneal injections of DNAase in dose of 1 mg/kg of body weight twice a day every day from day 3 to day 5 after the tumor cell transplantation.
- mice were administered with intraperitoneal injections of DNAase in dose of 1 mg/kg of body weight twice a day every day from day 3 to day 10 after the tumor cell transplantation.
- mice were administered with intraperitoneal injections of DNAase in dose of 1 mg/kg of body weight twice a day every day from day 3 to day 15 after the tumor cell transplantation.
- mice were administered with intraperitoneal injections of DNAase in dose of 1 mg/kg of body weight twice a day every day from day 3 to day 18 after the tumor cell transplantation.
- mice were administered with intraperitoneal injections of DNAase in dose of 1 mg/kg of body weight twice a day every day on 3,5,7,9,11,13,15 and 17 day after the tumor cell transplantation.
- mice were administered with intraperitoneal injections of DNAase in dose of 0,5 mg/kg of body weight four times daily every day from day 3 to day 10 after the tumor cell transplantation.
- the results was evaluated as animal survival on day 30 and day 50 after the tumor cell transplantation. The results are presented in the table 2.
- day 30 Animal survival on day 30 and day 50 after the tumor cell transplantation.
- day 50 (amount of alive/dead (amount of alive/dead Group animals in group animals in group) 1 0-7 0-7 2 0-6 0-6 3 3-3 0-6 4 5-1 3-3 5 6-0 6-0 6 0-6 0-6 7 4-2 1-5
- the presented data demonstrated that the therapy efficacy increases as the treatment time extends.
- the therapy efficacy is decrease if it is not uninterrupted. Multiple-dose administration is preferred.
- dornaze—alpha subcutaneous injections of dornaze—alpha were prescribed.
- the treatment began with administration of daily dose of 50 mkg/kg. Every consecutive day blood extracellular DNA level was measured and blood extracellular DNA was fractioned by electrophoresis. Once a week the primary tumor site and metastases were checked with X-rays and NMR-tomography. After initial 7 day period the dornaze-alpha daily dose has been increased up to 100 mkg/kg because of no changes in level and electrophoresis pattern of blood extracellular DNA and no reactions from primary site of the tumor and the metastases. Because of no changes after another 7 days the dosing has been increased up to 150 mkg/kg.
- the probes of patient's extracellular DNA taken before the treatment started and 21 days after the beginning the therapy were cloned by means a method which allowed to construct non amplificated plasmid libraries of blood extracellular DNA with representativeness up to one million of clones with the average size of 300-500 base pairs.
- DNA was treated by Eco RI restrictase during 3 hours or by Pfu polymerase (Stratagene) in presence of 300 mkM of all desoxynucleotidtriphosphates for sticky-ends elimination.
- the completed DNA was phosphorylated by polynucleotidkinase T4 (30U, 2 h.).
- the preparations were ligated to pBluescript plasmide (Stratagene), which had been digested with EcoRI or PvuII and dephosphorylated by phosphatase CIP (Fermentas) during 1 hour. 1 mkg of vector and 0,1-0,5 mkg of serum DNA were used.
- the ligated library was transformed into DH12S cells (Life Technologies) by meant of electroporator E. Coli porator (BioRad). 12-20 electroporation covets were used for the transformation of one library.
- the library serial dilutions of 10 ⁇ 4 , 10- 5 and 10 ⁇ 6 were cloned on 1,5% agar and LB media supplemented with 100 mkg/ml of ampicilline. In both cases the libraries represented 2-3*10 6 clones.
- Some class homologues are the products of cellular oncogenes. Fibroblast activation Involved into cancer invasion and metastasis. protein, cell surface protease Brain testican Reported in embryonic rhabdomyosarcoma. KRAB domain, Zn- Reported in early embryogenesis, finger protein. neuroblastoma, Ewing sarcoma, T-cell lymphoma, linked with acquisition of drug resistance in lung cancer. Melanoma Antigen expressed in melanoma cells. associated antigen N-cadherin Involved into cancer invasion and metastasis.
- DNAase in doses which are sufficient for destroying extracellular blood DNA with size higher than 300 base pairs leads to disappearing of unique fragments of human genome from extracellular blood DNA, including those involved into development and maintenance of cancer cells malignant behavior. At the same time the tumor respond to applied therapy.
- Antibodies against DNA were isolated from the blood of patients with systemic lupus erythematosus according to method of Shuster. A. M. (Shuster A. M. et. al., Science, v. 256, 1992, pp. 665-667). Such anti-DNA antibodies could not only bind DNA but also hydrolyze it. Human recombinant DNAase I(Genetech) was used.
- mice bearing Erlich carcinoma and being have got intravenous injection of human anti-DNA antibodies (Ig G) in dose of 200 mkg per animal on day 3 after the carcinoma transpalantation Mice also have been administered with DNAase in dose 0, 5 mg/kg 4 times intraperitonealy a day from day 3 to day 7 after the tumor transplantation.
- Ig G human anti-DNA antibodies
- Ig G human non-specific immunoglobulin
- TGI tumor growth inhibition on day 7 after the tumor cell transplantation
- the tumor volume on day 7 after tumor transplantation TABLE 3 Group Tumor volume T % 1 105 +/ ⁇ 12 — 2 25 +/ ⁇ 5 ⁇ 75% 3 37 +/ ⁇ 6 ⁇ 66% 4 35 +/ ⁇ 7 ⁇ 67%
- the respective enzyme was added to a sample of patient's plasma and incubated for 45 minutes at 37° C. 45 minutes later the reaction has being stopped and isolation and electrophoretic fractioning with densitometry of blood extracellular DNA have being performed.
- C57B1 mice have been inoculated with high metastatic or low metastatic strain of LLC tumor.
- On the 9 th day after the inoculation animals were euthanized and pool of their blood plasma was collected. The summary fraction of extracellular blood plasma DNA was kept in phosphate buffer at t ⁇ 20° C.).
- mice inoculated with low metastatic strain of LLC were included in the experiment.
- mice grafted by low metastatic LLC strain were subjected to twice repeated intravenous administration (on 7 and 8 day after inoculation) of summary fraction of extracellular DNA from mice grafted by high metastatic strain (before the administration 0,05 mkg of DNA have been dissolved in 500 mkl of fresh heparinized blood)
- mice grafted by low metastatic LLC strain were subjected to twice repeated intravenous administration (on 7 and 8 day after inoculation) of summary fraction of extracellular DNA from mice grafted by high metastatic strain (before the administration 0,05 mkg of DNA have been dissolved in 500 mkl of fresh heparinized blood). Before the administration the sample with DNA has been mixed with 10 mkg of hydrolytic anti-DNA antibodies.
- mice grafted by low metastatic LLC strain were subjected to twice repeated intravenous administration (on 7 and 8 day after inoculation) of summary fraction of extracellular DNA of mice graft by high metastatic strain (before the administration 0,05 mkg of DNA have been dissolved in 500 mkl of fresh heparinized blood).
- Ricin is the representative of RIP-toxins family (proteins inactivating ribosomes) which widely used for immunotoxins' development. In addition to its capability to inactivate ribosomes these proteins also can deadenilate and hydrolyze DNA.
- unit A of the type II RIP toxin should be delivered into cell by unit B.
- chain A is not toxic, however polyadeninglicosidase activity of chain A can be used for destruction of DNA circulating in blood.
- mice grafted by low metastatic LLC strain were subjected to twice repeated intravenous administration (on 7 and 8 day after inoculation) of summary fraction of extracellular DNA from mice grafted by high metastatic strain (before the administration 0,05 mkg of DNA have been dissolved in 500 mkl of fresh heparinized blood).
- the DNA sample was enzymatically methylated before the administration.
- mice grafted by low metastatic LLC strain were subjected to twice repeated intravenous administration (on 7 and 8 day after inoculation) of summary fraction of extracellular DNA of mice graft by low metastatic strain
- mice grafted by low metastatic LLC strain were subjected to twice repeated intravenous administration (on 7 and 8 day after inoculation) of summary fraction of extracellular DNA of mice grafted by high metastatic strain (before the administration 0,05 mkg of DNA have been dissolved in 500 mkl of fresh heparinized blood).
- N cp The number of lung metastases
Abstract
Description
- The invention is related to medicine and veterinary and can be used for treating in the main of solid tumors.
- Populations of tumor cells, which develop in the body of the patient, have very high genetic variability, which exceeds the same of healthy cells. Genetic variability of cancer cell populations allows their generate phenotypes, which are insensitive for immune and morphogenetic control and which have ability to invasion and metastasis and are insensitive for cancer therapy. It is considered that selection and clonal expansion of cancer cells underlie biological and clinical <<progression>> of tumors. That's why the strategy of modern cancer therapy is based on the principle of destroying of cancer cells clones in the body of patient with chemotherapy, radiotherapy, immunotherapy, biotherapy, surgical methods and the combination thereof.
- Chemotherapy, radiotherapy, biotherapy and more recently the immunotherapy are the most-used non-surgical methods of treating of cancer diseases and its are meant for destruction, damage or inactivation of cancer cell intracellular DNA.
- The chemotherapy approach is based on well known compounds: for platinum preparations see “Molecular mechanisms involved in cisplatin cytotoxicity”. Jordan P, Carmo-Fonseca M, Cell Mol Life Sci August 2000 v 57: pp. 1229-35, for anthracycline antibiotics, see “Daunorubicin and doxorubicin, anthracycline antibiotics, a physicochemical and biological review”, Aubel-Sadron G, Londos-Gagliardi D, Biochimie May 1984 v. 66: pp. 333-52, for alkylating agents, see “An overview of cyclophosphamide and ifosfamide pharmacology” and for podophyllotoxins, see “Podophyllotoxins: current status and recent developments”, Damayanthi Y, Lown J W, Curr Med Chem June 1998 5: v 3 205-52.
- Methods of radioimmunotherapy which allow to irradiate the intracellular DNA of cancer cells' nuclei by alpha particles from alpha-emitters which are specially delivered into cancer cells for increasing the effect on intracellular DNA, obtain the recognition, see “Targeted alpha therapy: evidence for potential efficacy of alpha-immunoconjugates in the management of micrometastatic cancer” Allen B J, Australas Radiol November 1999 v. 43: pp 480-6.
- There are biotherapeutic and immunotherapeutic methods, which meant for induction of apoptosis of cancer cells—the process of death of cancer cell, which starts with the activation of intracellular nucleases with following degradation of intracellular DNA of the tumor cell, for example by means administration of genotherapeutic constructions, which consists of genes inducing apoptosis, see “Phase I trial of adenovirus-mediated p53 gene therapy for recurrent glioma: biological and clinical results” Lang F F, Bruner J M, Fuller G N, Aldape K, Prados M D, Chang S, Berger M S, McDermott M W, Kunwar S M, Junck L R, Chandler W, Zwiebel J A, Kaplan R S, Yung W K, J Clin Oncol July 2003 1 21:13 2508-18, or genes coding the factors which activate the nucleases triggering apoptosis, see “Adenovirus-mediated transfer of caspase-8 augments cell death in gliomas: implication for gene therapy.” Shinoura N, Koike H, Furitu T, Hashimoto M, Asai A, Kirino T, Hamada H, Hum Gene Ther May 2000 20 11:8 1123-37, or by meant of anticancer vaccines, see “Vaccine-induced apoptosis: a novel clinical trial end point?” Amin S, Robins R A, Maxwell-Armstrong C A, Scholefield J H, Durrant L G, Cancer Res June 2000 60: 3132-6.
- Use of endonuclease Endo SR for treatment cancer diseases by moode of intracellular delivery it into target cells is known, U.S. Pat. No. C, 6,455,250.
- From methods listed above, the chemotherapy with the Etopozide-4-Demetilpipodophylotoxe(4,6-O—R)-etiliden)-b-D-glycopiranozid was selected by us as a prototype.
- Topoizomeraze II is essential cell enzyme which regulates many aspects of DNA function. The enzyme is responsible for interconversion of different topological forms of intracellular DNA by means of generation of transitory breaks of double-stranded DNA. Etopozide, as Topoizomeraze II inhibitor, increases intracellular level of “broken DNA—Topoizomeraze II” complexes.
- The result of this drug influence is the accumulation of the double-stranded intracellular DNA breaks which lead to cell death, see “Topoisomerase II as a target for anticancer drugs: when enzymes stop being nice”. Fortune J M, Osheroff N., Prog Nucleic Acid Res Mol Biol 2000, v. 64: pp. 221-53.
- The drawback of the method-prototype as well as any other well-known methods, is their low efficacy. It is explained by following. Method-prototype as well as other known methods implies the cancer cell and mostly the intracellular DNA as a therapeutic target.
- The experience of such therapy testifies that:
- because of high genetic variability, cancer cells become, as a rule no sensitive to the therapy before they are adequately eliminated by means of the using method;
- intracellular DNA is a difficult-to-approach target, and it leads to necessity of high-dosing antineoplastic chemotherapy and/or necessity of complicated delivery systems.(Drug Delivery Systems) As well, method-prototype which makes provision for influence on intracellular DNA of cancer cells also damages DNA of healthy cells and that's reason of it's high toxicity.
- The solving of the aim of development of highly efficient and low-toxic method of cancer therapy is the basis of this invention. According to the invention this problem is resolved by administrating into systemic circulation an agent which destroys blood extracellular DNA; and the said agent can be introduced in the dose which provide the alteration an electrophoretic profile of blood extracellular DNA, which could be detectable by puls-gelelectrophoresis; the agent can be introduced in doses and regime schedule providing a plasma hydrolytic activity, measured in blood plasma which exceeds 150 Kuntz units/liter of blood plasma, and this level can be supported for more than 12 hours within 24 hours in total; the treatment can be carried out continuously no less than 48 hours; DNAase enzyme can be used as agent to destroy extracellular blood DNA; in particular bovine pancreatic DNAase can be introduced parenterally in doses ranged from 50 000 Kunitz units/day till 25 000 000 000 Kunitz units/day per day every day during period of 5-360 days, in particular recombinant human DNAase (dornase—alpha) can be parenterally introduced in doses ranged from 0,15 mg/day till 500 mg/day per day every day during period of 5-360 days; the treatment can be life long; in addition an agent which bound extracellular DNA can be introduced to the systematic circulation; as this agent anti-DNA antibodies can be used; in addition an agent which modify the chemical structure and/or the conformation and/or the degree of polymerization and/or association with proteins and/or lipids and/or ribonucleic acids of extracellular DNA of the blood can be introduced to the systemic circulation; ribonuclease enzyme can be used as such agent; in particular the extracellular nuclease of Serratia Mercenses can be used.
- The fact of circulation of extracellular DNA in blood of cancer patients was described in a number of papers, see (P. Anker et al., Clinica Chimica Acta, v. 313, 2001, pp. 143-146; Fedorov N. A. et. al., Bull. Exp. Biol. Med., v 102,1986, pp. 281-283). In patent U.S. Pat. No. C, 5,952,170 the determination of extracellular DNA in blood for diagnostics and prognosis of clinical course of a malignant disease was described. In patents U.S. Pat. No. C, 6,465,177 and U.S. Pat. No. C, 6,156,504 there was described the use of extracellular DNA of blood for definition of mutations in oncogenes and microsatellic fragments of genes. Usage of blood extracellular DNA for studying the genome instability in tumors and application of the results for diagnostics, monitoring and prognosing were also disclosed.
- Besides, there was no systematic analysis of extracellular blood DNA spectrum and it's biological role before now. Published data of investigation of blood extracellular DNA performed without polymerase chain reaction (PCR) method were not found. PCR can pervert the pattern of blood extracellular DNA because of specificity of primers which are used for amplification. Until recent times the genetic analysis of extracellular blood DNA was mainly carried out by PCR or blot-hybridization and it was directed to the studying of changes in certain fragments of genome, for example in micro satellites and separate genes during malignant process (Sanchez-Cespedes M., et al., Ann Oncol, 1998, v 9(1), pp. 113-116; Sozzi G., et al., Clin Can Res, 1999, v. 5(10), pp. 2689-2692; Chen X. Q., et al., Nat Med, 1996, v. 2(9), pp. 1033-1035).
- Thus, in source of available data, there are no knowledge about genetic repertoire of extracellular blood DNA of cancer patients, biological role of extracellular blood DNA in oncopatology and potential therapeutic effects of destroying or inactivation for treating these diseases and according to aforesaid the invention conformances to requirements of “novelty” criteria (N).
- As the applicant established, the extracellular blood DNA of cancer patients contains the unique quantitative and qualitative repertoire of genes and regulating genetic elements which great differ from the repertoire of DNA which is described in human genome. In contrast to inracellular DNA the extracellular DNA of cancer patients contains mainly unique human genes, including genes which are involved in development and maintenance of malignant behavior of cancer cells.
- It is shown that extracellular DNA of blood of cancer patients contributes the malignant growth.
- Destroying, modification and binding of extracellular blood DNA slows down the malignant growth. Such intervention is very useful as independent therapy and it also increases the effectiveness of traditional methods of treatment.
- Aforesaid new characteristics of this invention are based on new ideas about mechanisms of oncology diseases. In this way this method conformances to requirements of “invention step” criteria (IS).
- As set for below the invention has been explained by detailed description of embodiments without references to drawings.
- The inventive method is realized as followed:
- Materials and Methods.
- The following agents were used which destroys extracellular blood DNA: bovine pancreatic DNAase (Sigma and Samson Med), recombinative human DNAase 1 (Dornase alpha; Genetech), Serratia Mercenses extracellular nuclease. The solutions of DNA-se for administration was made by dissolving of mother solutions of DNA-se in sterile phosphate buffer just before administration.
- Extracellular DNA from blood plasma was isolated as follows: fresh plasma (no more than 3-4 hours after sampling) was centrifuged on Ficoll-PlaquePlus (Amersham-Pharmacia) during 20 minutes at 1500 g at room temperature. ½ of plasma was detached, not affecting the rest of cells on the Ficoll pillow, and further centrifuged at 10000 g during 30 min for separation from cell fragments and debris. Supernatant was detached, without affecting of the sediment, and was toped up to 1% of sarkosil, 50
M M tris-HCl, pH 7,6, 20M M EDTA, 400M M NaCl, and than mixed with equal volume of phenol-chloroform(1:1) mixture. The prepared emulsion was incubated during 2 hours at t=65° C., then phenol-chloroform mixture was separated by centrifuging (500 g during 20 minutes, room temperature). - The procedure of deproteinisation with phenol—chlorophorm mixture was repeated 3 times, and then the water phase was processed with chloroform and diethyl ether. Separation from organic solvents was made by centrifugation at 5000 g during 15 minutes). Then equal volume of izopropanol was added to resulting aqueous phase and the mixture was incubated overnight at 0° C. After sedimentation the nucleic acids were separated by centrifugation at 10000 g during 30 minutes. The sediment of nucleic acids was dissolved in of 10
M M tris-HCl buffer, pH 7, 6 with 5M M EDTA, and inflicted to the CsCl gradient (1M, 2.5M, 5.7M) in test-tube for rotor SW60Ti. The volume of DNA solution was 2 ml, volume of each step of CsCl was 1 ml. Ultracentrifugation was conducted in L80-80 (Beckman) centrifuge during 3 hours at 250000 g. DNA was collected from the surface of each gradient step into fractions. These fractions were dialyzed during 12 hours (t=4° C.). Presence of DNA in fractions was determined by agar electrophoresis and DNA was visualized by ethidium bromide staining. The amount of DNA was determined with specrophotometer (Beckman DU70) in cuvet (100 mcl) at wavelength of 220-230 nm. - Mice Lewis lung carcinoma and Erlich carcinoma were used in experiments. Cells were cultivated in RPMI-1640 medium with 10% calf serum and 1% penicillin-streptomycin in atmosphere of 5% CO2.
- For tumors inoculation in mice the cells were cultivated till monolayer is formed, then detached with tripsin-EDTA buffer. The cells were washed 3 times by centrifuging in phosphate buffer and then resuspended up to 0, 5*107/ml concentration in the same buffer. The cell viability was determined with metylene blue staining in haemocitometer. Cells suspensions with no less than 95% of living cell were used for transplantation.
- C57B1 mice and white randomly breeded mice from “Rappolovo” animal house were used. Weight of animals was 24-26 g. 6-7animals were kept in one cage on a standard diet without limitation of water. LLC cells in dose 5*105 per mice in 0,1 ml of phosphate buffer were transplanted into thigh soft tissues. Erlich tumors were transplanted by administration of 0,2 ml of 10% cell suspension in physiological solution.
- In some experiments level of extracellular DNA in blood plasma was quantified. DNA was isolated according to aforesaid protocol. The DNA level was measured with PicoGreen kit. Electrophoresis of extracellular blood DNA was performed with 1% agar gel. DNA was visualized with etidium-bromide solution. The levels of high molecular DNA fraction (more than 300 base pairs) were determined by densitometry. Lambda phage DNA, digested with EcoRI and HindIII was used as electrophoresis marker.
- Recombinant human DNAase 1 (Genentech) was used.
- 1 group: 10 mice bearing Erlich carcinoma was used as control. The mice were injected with 0,2 ml of phosphate buffer intraperitoneally twice a day every day from day 3 to day 7 after the tumor cell transplantation.
- 2 group: 10 mice bearing Erlich carcinoma were introduced with intraperitoneal injections of DNAase in dose of 1 mg/kg of body weight in 0,2 ml of phosphate buffer four times daily every day from day 3 to day 7 after the tumor cell transplantation.
- 3 group: 10 mice bearing Erlich carcinoma were administered with intraperitoneal injections of DNAase in dose of 0,5 mg/kg of body weight in 0,2 ml of phosphate buffer four times daily every day from day 3 to day 7 after the tumor cell transplantation.
- 4 group: 10 mice bearing Erlich carcinoma were administered with intraperitoneal injections of DNAase in dose of 0,1 mg/kg of body weight in 0,2 ml of phosphate buffer four times daily every day from day 3 to day 7 after the tumor cell transplantation.
- 5 group: 10 mice bearing Erlich carcinoma were administered with intraperitoneal injections of DNAase in dose of 0,05 mg/kg of body weight in 0,2 ml of phosphate buffer four times daily every day from day 3 to day 7 after the tumor cell transplantation. The results were evaluated as tumor Growth Inhibitory Index(GII) (%) at the last day of DNAase injections. The quantification of blood plasma extracellular DNA and it's electrophoretic qualification were also performed.
- The results are presented in the table 1.
- Tumor size, extracellular DNA level and extracellular DNA electrophoresis profile on day 7 after tumor transplantation.
TABLE 1 Presence of high Extracellular molecular fractions Tumor Inhibition DNA level, of extracellular Group volume (%) (ng/ml) DNA Control 98 +/− 14 — 104.8 100%* 1 mg/kg\ 23 +/− 6 76% 38.3 0 0.5 mg/kg 32 +/− 6 67% 55.1 25% 0.1 mg/kg 58 +/− 12 37% 78.0 70% 0.05 mg/kg 87 +/− 11 10% 98.7 100%
*The control group electrophoretic density were considered as 100%.
- The presented data demonstrated that sufficiently high doses of DNAase 1 are needed to achieve the better therapeutic effect.
- Recombinant human DNAase I (Genentech) was used.
- 5 groups of mice bearing LLC were used.
- 1 group-7 mice-the control.
- 2 group-6 mice were administered with intraperitoneal injections of DNAase in dose of 1 mg/kg of body weight twice a day every day from day 3 to day 5 after the tumor cell transplantation.
- 3 group-6 mice were administered with intraperitoneal injections of DNAase in dose of 1 mg/kg of body weight twice a day every day from day 3 to day 10 after the tumor cell transplantation.
- 4 group-6 mice were administered with intraperitoneal injections of DNAase in dose of 1 mg/kg of body weight twice a day every day from day 3 to day 15 after the tumor cell transplantation.
- 5 group-6 mice were administered with intraperitoneal injections of DNAase in dose of 1 mg/kg of body weight twice a day every day from day 3 to day 18 after the tumor cell transplantation.
- 6 group-6 mice were administered with intraperitoneal injections of DNAase in dose of 1 mg/kg of body weight twice a day every day on 3,5,7,9,11,13,15 and 17 day after the tumor cell transplantation.
- 7 group-6 mice were administered with intraperitoneal injections of DNAase in dose of 0,5 mg/kg of body weight four times daily every day from day 3 to day 10 after the tumor cell transplantation. The results was evaluated as animal survival on day 30 and day 50 after the tumor cell transplantation. The results are presented in the table 2.
- Animal survival on day 30 and day 50 after the tumor cell transplantation.
TABLE 2 day 30 day 50 (amount of alive/dead (amount of alive/dead Group animals in group animals in group) 1 0-7 0-7 2 0-6 0-6 3 3-3 0-6 4 5-1 3-3 5 6-0 6-0 6 0-6 0-6 7 4-2 1-5 - The presented data demonstrated that the therapy efficacy increases as the treatment time extends. The therapy efficacy is decrease if it is not uninterrupted. Multiple-dose administration is preferred.
- 54-years-old man has been admitted to the hospital with diagnosis of lung carcinoma.
- By patient's agreement, due to lack of any available treatment modality, subcutaneous injections of dornaze—alpha were prescribed. The treatment began with administration of daily dose of 50 mkg/kg. Every consecutive day blood extracellular DNA level was measured and blood extracellular DNA was fractioned by electrophoresis. Once a week the primary tumor site and metastases were checked with X-rays and NMR-tomography. After initial 7 day period the dornaze-alpha daily dose has been increased up to 100 mkg/kg because of no changes in level and electrophoresis pattern of blood extracellular DNA and no reactions from primary site of the tumor and the metastases. Because of no changes after another 7 days the dosing has been increased up to 150 mkg/kg. Two days after the first injection of the preparation in dose 150 mkg/kg the material recession (more than 50%) of the blood extracellular DNA fraction with the size more than 300 base pairs has been observed although total amount of extracellular DNA has not been greatly decreased (less than 20%). During the next 4 days the patient's general condition has noticeably improved and on day 7 of this cycle of therapy 25%-decreasing of primary tumor lesion size and signs of regression of two bone metastases have been shown by NMR-scanning and X-ray examination. The probes of patient's extracellular DNA taken before the treatment started and 21 days after the beginning the therapy were cloned by means a method which allowed to construct non amplificated plasmid libraries of blood extracellular DNA with representativeness up to one million of clones with the average size of 300-500 base pairs. The DNA which have been isolated with aforesaid protocol was additionally deproteinizated with proteinase K (Sigma) at t=65 C for the removing of firm-binded proteins. After the deproteinization and single-stage treatment of phenol-chloroform mixture (t=65° C.) DNA was precipitated overnight with 2,5 volumes of ethanol. Then DNA was treated by Eco RI restrictase during 3 hours or by Pfu polymerase (Stratagene) in presence of 300 mkM of all desoxynucleotidtriphosphates for sticky-ends elimination. The completed DNA was phosphorylated by polynucleotidkinase T4 (30U, 2 h.). The preparations were ligated to pBluescript plasmide (Stratagene), which had been digested with EcoRI or PvuII and dephosphorylated by phosphatase CIP (Fermentas) during 1 hour. 1 mkg of vector and 0,1-0,5 mkg of serum DNA were used. The process of ligation was conducted with Rapid Legation Kit (Roche) during 10 hours at T=16° C. The volume of this mixture was 50 mkl. The ligated library was transformed into DH12S cells (Life Technologies) by meant of electroporator E. Coli porator (BioRad). 12-20 electroporation covets were used for the transformation of one library. The library serial dilutions of 10−4, 10-5 and 10−6 were cloned on 1,5% agar and LB media supplemented with 100 mkg/ml of ampicilline. In both cases the libraries represented 2-3*106 clones.
- Analysis of 96 randomly selected clones with the size 300-1000 base pairs from the “before treatment” library showed that 55 from 96 clones were the unique sequences of human DNA. For the 15 sequences from 55 the gene function or corresponding gene product were identified with HumanGeneBank.
Gene or Reported role in cancerogenesis and cancer corresponding progression protein product G-protein coupled Key role in neoplastic transformation, apoptosis receptor protein inhibition, hormone independence and metastasis Snf2 coupled CBP Transcription activator, reported in synovial activator (SCARP) sarcoma and leukemia. SRY-box containing Transcription modulator expressed in gene embryogenesis. Reported in medulloblastoma, gonadal tumors, highly metastatic melanoma. Tyrosine kinase Key role in cancer cell regulation network. Some class homologues are the products of cellular oncogenes. Fibroblast activation Involved into cancer invasion and metastasis. protein, cell surface protease Brain testican Reported in embryonic rhabdomyosarcoma. KRAB domain, Zn- Reported in early embryogenesis, finger protein. neuroblastoma, Ewing sarcoma, T-cell lymphoma, linked with acquisition of drug resistance in lung cancer. Melanoma Antigen expressed in melanoma cells. associated antigen N-cadherin Involved into cancer invasion and metastasis. Interleukin 7 Proposed essential autocrine - paracrine growth factor for many cancers DEAD Box RNA Expressed in highly proliferating and cancer helicase-like protein cells. Lipin-1 Involved into cancer cell response to cytotoxic drugs. Dynein Participate in p53 intracellular traffic, reported in prostate cancer and hepatocellular carcinoma. Ramp protein Reported in human embryonic carcinoma - Analysis of 100 clones selected randomly from the “21 day after treatment” library showed that more than 90% sequences of clones represented short fragments of repetitive DNA of human genome with dominance of alpha-satellite DNA.
- Hence the use of DNAase in doses which are sufficient for destroying extracellular blood DNA with size higher than 300 base pairs leads to disappearing of unique fragments of human genome from extracellular blood DNA, including those involved into development and maintenance of cancer cells malignant behavior. At the same time the tumor respond to applied therapy.
- The treatment of malignant low differentiated lymphoma invading the spleen and portal vien and metastases in the liver.
- 49-years-old woman has been admitted to the hospital with the fever (39 C), progressive jaundice, liver failure and being under suspicion of acute hepatitis suffering. During the inspection malignant lymphoma with the difflusely defeats of spleen and gates of liver and multiply metastases in liver were revealed. By patient's agreement, due to the lack of any specific treatment and because of progressing of the disease, intravenous injections of bovine pancreatic DNAase were prescribed. Twice a day measuring of level of blood extracellular DNA and it's electrophoretic fractioning were conducted. During the first day 500000 units of enzyme were administered as 26-hour infusions. Later this dose was increased by 1 000 000 units per day. When the dose was 5500000 units daily the 50% decrease of blood extracellular DNA and disappearance of fraction of DNA with size more than 300 base pairs were noted. As the continued DNA infusions at 5500000 units per day were being performed the patient's general condition was being improved, fever and jaundice disappeared, biochemical indexes of blood taken a turn to the better. Control Doppler examination which has been made at day 20 after the beginning of the treatment showed significant reduction (more than 40%) of lesion in the spleen and disappearance of more than half of all metastatic sites in the liver. The woman was moved to another hospital for conducting chemotherapy.
- Hence the use of DNAase in doses which are sufficient for destroying extracellular DNA of blood with size higher than 300 base pairs leads to tumor regression according to the inventive method.
- The study of influence of polyclonal serum containing the antibodies against DNA on the growth of Erlich carcinoma of in mice treating with DNAase.
- Antibodies against DNA were isolated from the blood of patients with systemic lupus erythematosus according to method of Shuster. A. M. (Shuster A. M. et. al., Science, v. 256, 1992, pp. 665-667). Such anti-DNA antibodies could not only bind DNA but also hydrolyze it. Human recombinant DNAase I(Genetech) was used.
- 1 group-7 mice bearing Erlich carcinoma—control
- 2 group-6 mice bearing Erlich carcinoma and being have got intravenous injection of human anti-DNA antibodies (Ig G) in dose of 200 mkg per animal on day 3 after the carcinoma transpalantation Mice also have been administered with DNAase in dose 0, 5 mg/kg 4 times intraperitonealy a day from day 3 to day 7 after the tumor transplantation.
- 3 group-6 mice bearing Erlich carcinoma and being have got intravenous injection of human non-specific immunoglobulin (Ig G) in dose of 200 mkg per animal on day 3 after the carcinoma transpalantation. Mice also have been administered with DNAase in dose 0, 5 mg/kg 4 times intraperitonealy a day from day 3 to day 7 after the tumor transplantation.
- 4 group-6 mice bearing Erlich carcinoma and being have got intraperitoneal treatment of human DNAase in dose of 0,5 mg/kg 4 times intraperitonealy a day from day 3 to day 7 after the tumor transplantation.
- The effect was evaluated as the tumor growth inhibition on day 7 after the tumor cell transplantation (TGI, evaluated in percents). The results are presented in the table 3.
- The tumor volume on day 7 after tumor transplantation
TABLE 3 Group Tumor volume T % 1 105 +/− 12 — 2 25 +/− 5 ˜75% 3 37 +/− 6 ˜66% 4 35 +/− 7 ˜67% - The presented data demonstrated that the combined therapy with DNAase and the agent binding blood exracellular DNA has more noticeable antitumor effect.
- The study of degradation kinetics of high molecular weight fraction (size more than 300 pairs of bases) of blood extracellular DNA of breast cancer patient in presence of: bovine pancreatic DNAase, Proteinase K and bovine pancreatic DNAase, Lipase and bovine pancreatic DNAase and extracellular desoxyrybonuclease Serratia Mercenses, which has ribonuclease activity and is as destroyed and modificating agent at the same time.
- The respective enzyme was added to a sample of patient's plasma and incubated for 45 minutes at 37° C. 45 minutes later the reaction has being stopped and isolation and electrophoretic fractioning with densitometry of blood extracellular DNA have being performed.
- The results are presented in the table 4.
- Degradation kinetics of high molecular fraction
TABLE 4 Degradation of high molecular The way of working fraction, % Intact control 0 Proteinase K (0.1 mkg/ml) 0 Pancreatic lipase (0.1 mkg/ml) 0 Bovine pancreatic DNAase 25 (1 Kuntz Units/ml) Bovine pancreatic DNAase 35 (1 Kuntz Units\ml) + proteinase K (0.1 mkg\ml) Bovine pancreatic DNAase 40 (1 Kuntz Units/ml) + pancreatic lipase (0.1 mkg/ml) Extracellular desoxyribonuclease of Serratia 45 Mercenses (1 Kuntz Units/ml) - The presented data demonstrated that the combined therapy with DNAase and the agent modificating blood exracellular DNA binding with proteins, lipids and ribonucleic acids leads to more effective degradation of high molecular fraction (size more than 300 pairs of bases) of blood extracellular DNA
- The study of the influence of different methods of destroying extracellular DNA on it's pathogenic properties.
- C57B1 mice have been inoculated with high metastatic or low metastatic strain of LLC tumor. On the 9 th day after the inoculation animals were euthanized and pool of their blood plasma was collected. The summary fraction of extracellular blood plasma DNA was kept in phosphate buffer at t=−20° C.).
- 7 groups of mice inoculated with low metastatic strain of LLC were included in the experiment.
- 1 group-6 mice grafted by low metastatic LLC strain.
- 2 group-6 mice grafted by low metastatic LLC strain and were subjected to twice repeated intravenous administration (on 7 and 8 day after inoculation) of summary fraction of extracellular DNA from mice grafted by high metastatic strain (before the administration 0,05 mkg of DNA have been dissolved in 500 mkl of fresh heparinized blood)
- 3 group-6 mice grafted by low metastatic LLC strain and were subjected to twice repeated intravenous administration (on 7 and 8 day after inoculation) of summary fraction of extracellular DNA from mice grafted by high metastatic strain (before the administration 0,05 mkg of DNA have been dissolved in 500 mkl of fresh heparinized blood). Before the administration the sample with DNA has been disinfected photochemically (by adding 1 mkM of methylen blue stain and exposure of red light during 10 min (˜60 000 lux).
- 4 group-6 mice grafted by low metastatic LLC strain and were subjected to twice repeated intravenous administration (on 7 and 8 day after inoculation) of summary fraction of extracellular DNA from mice grafted by high metastatic strain (before the administration 0,05 mkg of DNA have been dissolved in 500 mkl of fresh heparinized blood). Before the administration the sample with DNA has been mixed with 10 mkg of hydrolytic anti-DNA antibodies.
- 5 group-6 mice grafted by low metastatic LLC strain and were subjected to twice repeated intravenous administration (on 7 and 8 day after inoculation) of summary fraction of extracellular DNA of mice graft by high metastatic strain (before the administration 0,05 mkg of DNA have been dissolved in 500 mkl of fresh heparinized blood). Before the administration 1 mkg of the fragment A of the plant toxin Ricin was added to the sample and the mixture was incubated during 1 hour at =37° C. Ricin is the representative of RIP-toxins family (proteins inactivating ribosomes) which widely used for immunotoxins' development. In addition to its capability to inactivate ribosomes these proteins also can deadenilate and hydrolyze DNA. To realize of the toxic effect the unit A of the type II RIP toxin should be delivered into cell by unit B. In absence of subunit B chain A is not toxic, however polyadeninglicosidase activity of chain A can be used for destruction of DNA circulating in blood.
- 6 group-6 mice grafted by low metastatic LLC strain were subjected to twice repeated intravenous administration (on 7 and 8 day after inoculation) of summary fraction of extracellular DNA from mice grafted by high metastatic strain (before the administration 0,05 mkg of DNA have been dissolved in 500 mkl of fresh heparinized blood). The DNA sample was enzymatically methylated before the administration. (I. Muiznieks et. al., FEBS Letters, 1994, v. 344, pp. 251-254).
- 7 group-6 mice grafted by low metastatic LLC strain were subjected to twice repeated intravenous administration (on 7 and 8 day after inoculation) of summary fraction of extracellular DNA of mice graft by low metastatic strain
- 8 group-6 mice grafted by low metastatic LLC strain were subjected to twice repeated intravenous administration (on 7 and 8 day after inoculation) of summary fraction of extracellular DNA of mice grafted by high metastatic strain (before the administration 0,05 mkg of DNA have been dissolved in 500 mkl of fresh heparinized blood). The sample with DNA was incubated with 200 ng/ml of dornase alpha during 30 minutes at T=37° C. before the administration.
- The number of lung metastases (N cp) was evaluated on day 15 after the inoculation.
- The results are presented in the table 5.
- The number of lung metastases on day 15 after the tumor inoculation subjectto the extracellular DNA destruction method.
TABLE 5 Group N cp 1 12.0 2 22.5 3 14.1 4 15.5 5 15.1 6 12.3 7 13.3 8 13.5 - Hence blood extracellular DNA of mice bearing highly malignant tumor strain increases metastasis of less malignant tumor. Destruction, binding and modification of blood extracellular DNA suppress that process according to the inventive method.
- For the realization the methods there were used well-known materials and equipment manufactured in plant conditions and according to aforesaid the invention conformances to requirements of “industrial applicability” criteria (IA).
Claims (14)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/708,914 US8710012B2 (en) | 2003-07-14 | 2010-02-19 | Method for treating oncological diseases |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/RU2003/000304 WO2005007187A1 (en) | 2003-07-14 | 2003-07-14 | Method for treating oncological, virulent and somatic diseases, method for controlling treatment efficiency, pharmaceutical agents and composition for carrying out said treatment |
RU03/00304 | 2003-07-14 | ||
RU2004108061/14A RU2269356C2 (en) | 2003-07-14 | 2004-03-12 | Method for treating oncological patients |
RU2004108061 | 2004-03-12 | ||
PCT/RU2004/000261 WO2005004903A1 (en) | 2003-07-14 | 2004-07-01 | Method for treating oncological diseases |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/708,914 Continuation-In-Part US8710012B2 (en) | 2003-07-14 | 2010-02-19 | Method for treating oncological diseases |
Publications (2)
Publication Number | Publication Date |
---|---|
US20060233780A1 true US20060233780A1 (en) | 2006-10-19 |
US7612032B2 US7612032B2 (en) | 2009-11-03 |
Family
ID=34067504
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/564,861 Active 2025-07-06 US7612032B2 (en) | 2003-07-14 | 2004-07-01 | Method for treating oncological diseases |
US10/564,615 Abandoned US20080004561A1 (en) | 2003-07-14 | 2005-01-27 | Method for Treating Oncological, Virulent and Somatic Diseases, Method for Controlling Treatment Efficiency, Pharmaceutical Agents and Composition for Carrying Out Said Treatment |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/564,615 Abandoned US20080004561A1 (en) | 2003-07-14 | 2005-01-27 | Method for Treating Oncological, Virulent and Somatic Diseases, Method for Controlling Treatment Efficiency, Pharmaceutical Agents and Composition for Carrying Out Said Treatment |
Country Status (6)
Country | Link |
---|---|
US (2) | US7612032B2 (en) |
EP (1) | EP1655036B1 (en) |
ES (1) | ES2427091T3 (en) |
PL (1) | PL1655036T3 (en) |
RU (1) | RU2269356C2 (en) |
WO (1) | WO2005004903A1 (en) |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090053200A1 (en) * | 2005-04-25 | 2009-02-26 | Dmitry Dmitrievich Genkin | Method for retarding unhealth manifestations brought by ageing of human beings |
WO2009134027A3 (en) * | 2008-04-30 | 2010-01-21 | Ajou University Industry-Academic Cooperation Foundation | Anti-nucleic acid antibody inducing cell death of cancer cells and composition for preventing or treating cancers comprising the same |
US20100061971A1 (en) * | 2006-11-28 | 2010-03-11 | Dmitry Dmitrievich Genkin | Method for Treating Human Diseases Associated With an Increased Deoxyribonucleic Acid Content in Extracellular Spaces of Tissues and a Medicinal Preparation for Carrying Out Said Method |
US20100150903A1 (en) * | 2003-07-14 | 2010-06-17 | Dmitry Dmitrievich Genkin | Method for Treating Oncological Diseases |
US20100303796A1 (en) * | 2003-07-14 | 2010-12-02 | Dmitry Dmitrievich Genkin | Method for Treating Systemic DNA Mutation Disease |
US20110189156A1 (en) * | 2003-07-14 | 2011-08-04 | Dmitry Dmitrievich Genkin | Method for Treating Systemic Bacterial, Fungal and Protozoan Infection |
AU2013265070B2 (en) * | 2012-05-21 | 2017-08-24 | London School Of Hygiene And Tropical Medicine | Therapeutic for treating Clostridium difficile infection |
WO2019212694A1 (en) * | 2018-04-30 | 2019-11-07 | Viktor Veniaminovich Tets | Tetz-proteins and prion-like proteins and associated methods |
US10617743B2 (en) | 2014-06-19 | 2020-04-14 | Cls Therapeutics Limited | Method to improve safety and efficacy of anti-cancer therapy |
US11701410B2 (en) | 2015-05-22 | 2023-07-18 | Cls Therapeutics Limited | Extracellular DNA as a therapeutic target in neurodegeneration |
US11905522B2 (en) | 2018-01-16 | 2024-02-20 | Cls Therapeutics Limited | Treatment of diseases by liver expression of an enzyme which has a deoxyribonuclease (DNase) activity |
US11918610B2 (en) | 2018-06-29 | 2024-03-05 | Viktor Veniaminovich Tets | Methods for diagnosis and treatment of type 1 diabetes |
Families Citing this family (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005007187A1 (en) * | 2003-07-14 | 2005-01-27 | Viktor Veniaminovich Tets | Method for treating oncological, virulent and somatic diseases, method for controlling treatment efficiency, pharmaceutical agents and composition for carrying out said treatment |
GB0713074D0 (en) | 2007-07-05 | 2007-08-15 | Univ London | A method of hyperpolarising a magnetic resonance agent |
WO2012071420A1 (en) * | 2010-11-23 | 2012-05-31 | The Trustees Of Columbia University In The City Of New York | Enzyme combinations to reduce brain tissue swelling |
EP3597665A1 (en) | 2011-04-01 | 2020-01-22 | Yale University, Inc. | Cell-penetrating anti-dna antibodies and uses thereof to inhibit dna repair |
WO2012166611A2 (en) | 2011-05-27 | 2012-12-06 | Immune Disease Institute, Inc. | Methods for treating and preventing neutrophil-derived net toxicity and thrombosis |
WO2013114373A1 (en) | 2012-02-01 | 2013-08-08 | Protalix Ltd. | Inhalable liquid formulations of dnase i |
WO2015134607A1 (en) | 2014-03-04 | 2015-09-11 | Yale University | Cell penetrating anti-guanosine antibody based therapy for cancers with ras mutations |
US10238742B2 (en) | 2014-06-25 | 2019-03-26 | Yale University | Cell penetrating nucleolytic antibody based cancer therapy |
WO2016108244A1 (en) | 2015-01-04 | 2016-07-07 | Protalix Ltd. | Modified dnase and uses thereof |
EP3368064B1 (en) | 2015-10-29 | 2021-05-19 | CLS Therapeutics Limited | Use of dnase to improve safety and efficacy of anti-cancer chemotherapy |
DE102016001407A1 (en) | 2016-02-09 | 2017-08-24 | Fresenius Medical Care Deutschland Gmbh | Blood treatment with inactivation of free nucleic acids |
CN109803687B (en) | 2016-06-15 | 2022-11-15 | 耶鲁大学 | Antibody-mediated autocatalytic targeted delivery of nanocarriers to tumors |
EP3351263A1 (en) | 2017-01-20 | 2018-07-25 | Universitätsklinikum Hamburg-Eppendorf | Pharmaceutical preparation for treating or preventing tissue adhesion |
WO2018197547A1 (en) | 2017-04-25 | 2018-11-01 | Lipoxen Technologies Limited | Methods of treating diseases related to net formation with parenteral administration of polysialylated dnase i |
AU2018318313A1 (en) | 2017-08-18 | 2020-03-19 | Neutrolis Inc.. | Engineered DNase enzymes and use in therapy |
AU2018332326A1 (en) | 2017-09-18 | 2020-05-07 | Santersus Ag | Method and device for purification of blood from circulating cell free DNA |
US10988746B2 (en) | 2018-10-08 | 2021-04-27 | Neutrolis, Inc. | Manufacturing and engineering of DNASE enzymes for therapy |
US11058724B2 (en) | 2018-10-08 | 2021-07-13 | Neutrolis, Inc. | Methods of using DNASE1-like 3 in therapy |
EP3864147A4 (en) | 2018-10-08 | 2022-07-06 | Neutrolis, Inc. | Engineering of dnase enzymes for manufacturing and therapy |
US11352613B2 (en) | 2019-02-04 | 2022-06-07 | Neutrolis, Inc. | Engineered human extracellular DNASE enzymes |
JP2023545740A (en) | 2020-10-07 | 2023-10-31 | プロタリクス リミテッド | Long-term active DNase |
EP4319788A1 (en) * | 2021-04-05 | 2024-02-14 | Victor Tets | Regulation of cells and organisms |
EP4340682A1 (en) * | 2021-05-21 | 2024-03-27 | Dmitry Dmitrievich Genkin | Immunomodulation of tumor microenvironment |
WO2023126672A1 (en) | 2021-12-27 | 2023-07-06 | Santersus Ag | Method and device for removal of circulating cell free dna |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5484589A (en) * | 1992-04-20 | 1996-01-16 | Rufeld, Inc. | Anti-viral methods using RNAse and DNAse |
CH686982A5 (en) | 1993-12-16 | 1996-08-15 | Maurice Stroun | Method for diagnosis of cancers. |
RU2099080C1 (en) * | 1994-02-11 | 1997-12-20 | Дмитрий Иванович Финько | Method for preventing from cancer development |
CA2248981C (en) | 1996-03-15 | 2009-11-24 | The Penn State Research Foundation | Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assays |
AU762972B2 (en) * | 1998-07-16 | 2003-07-10 | Research Development Foundation | Novel DNA-cleaving antitumor agents |
US6465177B1 (en) | 1998-10-26 | 2002-10-15 | John Wayne Cancer Institute | Detection of loss of heterozygosity in tumor and serum of melanoma patients |
AU4430701A (en) * | 2000-04-03 | 2001-10-15 | Antisoma Research Limited | Compounds for targeting |
-
2004
- 2004-03-12 RU RU2004108061/14A patent/RU2269356C2/en active
- 2004-07-01 WO PCT/RU2004/000261 patent/WO2005004903A1/en active Application Filing
- 2004-07-01 ES ES04748955T patent/ES2427091T3/en active Active
- 2004-07-01 EP EP04748955.4A patent/EP1655036B1/en not_active Not-in-force
- 2004-07-01 PL PL04748955T patent/PL1655036T3/en unknown
- 2004-07-01 US US10/564,861 patent/US7612032B2/en active Active
-
2005
- 2005-01-27 US US10/564,615 patent/US20080004561A1/en not_active Abandoned
Cited By (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9770492B2 (en) | 2003-07-14 | 2017-09-26 | Cls Therapeutics Limited | Method for treating systemic DNA mutation disease |
US20100303796A1 (en) * | 2003-07-14 | 2010-12-02 | Dmitry Dmitrievich Genkin | Method for Treating Systemic DNA Mutation Disease |
US9845461B2 (en) | 2003-07-14 | 2017-12-19 | Cls Therapeutics Limited | Method for treating oncological diseases |
US20100150903A1 (en) * | 2003-07-14 | 2010-06-17 | Dmitry Dmitrievich Genkin | Method for Treating Oncological Diseases |
US9463223B2 (en) | 2003-07-14 | 2016-10-11 | Cls Therapeutics Limited | Method for monitoring development of somatic mosaicism |
US9248166B2 (en) | 2003-07-14 | 2016-02-02 | Cls Therapeutics Limited | Method for treating oncological diseases |
US20110189156A1 (en) * | 2003-07-14 | 2011-08-04 | Dmitry Dmitrievich Genkin | Method for Treating Systemic Bacterial, Fungal and Protozoan Infection |
US9072733B2 (en) | 2003-07-14 | 2015-07-07 | Cls Therapeutics Limited | Method for treating systemic bacterial, fungal and protozoan infection |
US8431123B2 (en) | 2003-07-14 | 2013-04-30 | Cls Therapeutics Limited | Method for treating systemic bacterial, fungal and protozoan infection |
US8535663B2 (en) | 2003-07-14 | 2013-09-17 | Cls Therapeutics Limited | Method for treating delayed-type hypersensitivity |
US8710012B2 (en) | 2003-07-14 | 2014-04-29 | Cls Therapeutics Limited | Method for treating oncological diseases |
US8796004B2 (en) | 2003-07-14 | 2014-08-05 | Cls Therapeutics Limited | Method for treating systemic DNA mutation disease |
US8388951B2 (en) | 2003-07-14 | 2013-03-05 | Cls Therapeutics Limited | Method for treating systemic DNA mutation disease |
US8916151B2 (en) | 2005-04-25 | 2014-12-23 | Cls Therapeutics Limited | Method for treating a reduction of fertility |
US20090053200A1 (en) * | 2005-04-25 | 2009-02-26 | Dmitry Dmitrievich Genkin | Method for retarding unhealth manifestations brought by ageing of human beings |
US20100061971A1 (en) * | 2006-11-28 | 2010-03-11 | Dmitry Dmitrievich Genkin | Method for Treating Human Diseases Associated With an Increased Deoxyribonucleic Acid Content in Extracellular Spaces of Tissues and a Medicinal Preparation for Carrying Out Said Method |
US8871200B2 (en) | 2006-11-28 | 2014-10-28 | Cls Therapeutics Limited | Method for treating human diseases associated with an increased deoxyribonucleic acid content in extracellular spaces of tissues and a medicinal preparation for carrying out said method |
WO2009134027A3 (en) * | 2008-04-30 | 2010-01-21 | Ajou University Industry-Academic Cooperation Foundation | Anti-nucleic acid antibody inducing cell death of cancer cells and composition for preventing or treating cancers comprising the same |
US20110159569A1 (en) * | 2008-04-30 | 2011-06-30 | Ajou University IndustryAcademic Cooperation Found | Anti-nucleic acid antibody inducing cell death of cancer cells and composition for preventing or treating cancers comprising the same |
AU2013265070B2 (en) * | 2012-05-21 | 2017-08-24 | London School Of Hygiene And Tropical Medicine | Therapeutic for treating Clostridium difficile infection |
US10617743B2 (en) | 2014-06-19 | 2020-04-14 | Cls Therapeutics Limited | Method to improve safety and efficacy of anti-cancer therapy |
US11701410B2 (en) | 2015-05-22 | 2023-07-18 | Cls Therapeutics Limited | Extracellular DNA as a therapeutic target in neurodegeneration |
US11905522B2 (en) | 2018-01-16 | 2024-02-20 | Cls Therapeutics Limited | Treatment of diseases by liver expression of an enzyme which has a deoxyribonuclease (DNase) activity |
WO2019212694A1 (en) * | 2018-04-30 | 2019-11-07 | Viktor Veniaminovich Tets | Tetz-proteins and prion-like proteins and associated methods |
US11918610B2 (en) | 2018-06-29 | 2024-03-05 | Viktor Veniaminovich Tets | Methods for diagnosis and treatment of type 1 diabetes |
Also Published As
Publication number | Publication date |
---|---|
RU2269356C2 (en) | 2006-02-10 |
US20080004561A1 (en) | 2008-01-03 |
ES2427091T3 (en) | 2013-10-28 |
EP1655036A4 (en) | 2009-06-17 |
RU2004108061A (en) | 2005-09-27 |
WO2005004903A1 (en) | 2005-01-20 |
EP1655036B1 (en) | 2013-07-17 |
PL1655036T3 (en) | 2014-02-28 |
US7612032B2 (en) | 2009-11-03 |
EP1655036A1 (en) | 2006-05-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20060233780A1 (en) | Method for treating oncological diseases | |
US9845461B2 (en) | Method for treating oncological diseases | |
JP5307042B2 (en) | Pharmaceuticals and methods for treating human malignancies by depleting arginine | |
US20110312899A1 (en) | HDL Particles for Delivery of Nucleic Acids | |
Li et al. | Tumor microenvironment shapes colorectal cancer progression, metastasis, and treatment responses | |
JP5873808B2 (en) | Biologically effective molecules that affect cells and / or tumor cells infected with viruses, bacteria, parasites, and methods for applying the molecules | |
CN101965361A (en) | Biological materials and uses thereof | |
EP1666055A1 (en) | Method for treating oncological, virulent and somatic diseases, method for controlling treatment efficiency, pharmaceutical agents and composition for carrying out said treatment | |
Ghaemi et al. | Non-coding RNAs enhance the apoptosis efficacy of therapeutic agents used for the treatment of glioblastoma multiform | |
KR102132198B1 (en) | CRISPR system specific for TERT promoter mutation and use thereof | |
CN113227384A (en) | Pharmaceutical compositions, kits and methods for treating tumors | |
WO1997020579A2 (en) | Novel compounds and use | |
EP2497487B1 (en) | Method for treating diabetes and atherosclerosis associated with changes of qualitative and/quantitative composition of blood extracellular DNA | |
JP2005538709A (en) | Pharmaceuticals and methods for treating human malignancies by depleting arginine | |
Shahbaz et al. | Current advances in microbial-based cancer therapies | |
Okada et al. | Prevention of Lung Metastasis by Intra‐tumoral Injection of Cepharanthin and Staphylococcal Enterotoxin B in Transplantable Rat Osteosarcoma | |
WO2023077925A1 (en) | Drug for treating and/or preventing cancer and use thereof | |
CN100434437C (en) | Biological active peptides comprising isoleucyl-valyl-threonyl-asparaginyl-threonyl-threonine | |
Song et al. | Advances in anti-tumor based on various anaerobic bacteria and their derivatives as drug vehicles | |
Hess | Gene therapy: A brief review | |
AU2004217830B2 (en) | A recombinant constructed by a virus vector and a human tumor suppressor gene and its use | |
Davis | Investigating inhibition of porphyrin (heme) synthesis as a novel treatment option for prostate cancer | |
WAKABAYASHI et al. | Gene therapy of central nervous system tumors | |
Smith et al. | The Future of Therapeutic Options for Hereditary Angioedema | |
Ganesh et al. | A New Strategy For The Genetic Therapy Of Oral Cancer: An Update |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
AS | Assignment |
Owner name: CLS THERAPEUTICS LIMITED, UNITED KINGDOM Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:GENKIN, DMITRY DMITRIEVICH;TETS, VIKTOR VENIAMINOVICH;TETS, GEORGY VIKTOROVICH;REEL/FRAME:025358/0273 Effective date: 20100824 |
|
CC | Certificate of correction | ||
FEPP | Fee payment procedure |
Free format text: PAYOR NUMBER ASSIGNED (ORIGINAL EVENT CODE: ASPN); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY |
|
FPAY | Fee payment |
Year of fee payment: 4 |
|
FEPP | Fee payment procedure |
Free format text: PAT HOLDER NO LONGER CLAIMS SMALL ENTITY STATUS, ENTITY STATUS SET TO UNDISCOUNTED (ORIGINAL EVENT CODE: STOL); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY |
|
FPAY | Fee payment |
Year of fee payment: 8 |
|
MAFP | Maintenance fee payment |
Free format text: PAYMENT OF MAINTENANCE FEE, 12TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1553); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY Year of fee payment: 12 |